Previous studies carried out in this laboratory have provided data indicating that oral contraceptive steroids, particularly the synthetic estrogens, mestranol and ethinyl estradiol, are relatively strong promoters of hepatocarcinogenesis. It is now necessary to carry out studies designed to elucidate the mechanisms which may be involved. Such studies can be carried out at three levels, namely at the organismal, cellular and subcellular or molecular levels. This renewal application outlines a series of studies designed with the overall objective of obtaining new information on the mechanisms of promotion of hepatocarcinogenesis, in general, and synthetic estrogens, in particular, with emphasis on their effects at the organismal and cellular levels. The Specific Aims of this proposal are: (1) To study processes through which ethinyl estradiol may exert its promoting effects, by determining: (a-1) whether promotion by ethinyl estradiol involved chronic cholestasis leading to chronic hepatotoxicity and consequent restorative hyperplasia, and if so; (a-2) to evaluate whether elevated intraheptic bile acids resulting from chronic cholestasis are involved: (b) the extent to which promotion by ethinyl estradiol is mediated through estrogen receptor mechanisms; (2) To compare the mitogenic potency of acute exposure to several promoters of hepatocarcinogenesis on putative preneoplastic gamma glutamyl transpeptidase (GGT) foci and to determine whether differences in mitogenic potency are reflected in differences in promoting activity; (3) To confirm and extend previous results indicating that promotion by chronic treatment with synthetic estrogens results in increases in GGT lesion number, but not size, and to compare the phenotypic heterogeneity and 3H-thymidine labeling index of phenotypically altered lesions promoted by chronic phenobarbital or ethinyl estradiol treatment of female rats initiated with a low dose of diethylnitrosamine (DEN); (4) To determine whether the synthetic estrogens exert their promoting activity through direct effects on hepatocytes as indicated by enhanced focal growth of cultured rat hepatocytes isolated from DEN-initiated female rats and, if so, to determine whether among several possibilities, activated oxygen compounds or other free radicals are involved; and (5) To determine whether hepatocyte cell-cell interactions are altered during promotion.